Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1–associated membranous nephropathy
George Haddad, Johan M. Lorenzen, Hong Ma, Noortje de Haan, Harald Seeger, Christelle Zaghrini, Simone Brandt, Malte Kölling, Urs Wegmann, Bence Kiss, Gábor Pál, Péter Gál, Rudolf P. Wüthrich, Manfred Wuhrer, Laurence H. Beck, David J. Salant, Gérard Lambeau, Andreas D. Kistler
George Haddad, Johan M. Lorenzen, Hong Ma, Noortje de Haan, Harald Seeger, Christelle Zaghrini, Simone Brandt, Malte Kölling, Urs Wegmann, Bence Kiss, Gábor Pál, Péter Gál, Rudolf P. Wüthrich, Manfred Wuhrer, Laurence H. Beck, David J. Salant, Gérard Lambeau, Andreas D. Kistler
View: Text | PDF
Research Article Immunology Nephrology

Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1–associated membranous nephropathy

Abstract

Primary membranous nephropathy (pMN) is a leading cause of nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1–positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induced proteolysis of the 2 essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1 IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1 titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 were required to induce proteolysis of synaptopodin and NEPH1 by 2 distinct proteolytic pathways mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrated a mechanism by which aberrantly glycosylated IgG4 activated the lectin pathway and induced podocyte injury in primary membranous nephropathy.

Authors

George Haddad, Johan M. Lorenzen, Hong Ma, Noortje de Haan, Harald Seeger, Christelle Zaghrini, Simone Brandt, Malte Kölling, Urs Wegmann, Bence Kiss, Gábor Pál, Péter Gál, Rudolf P. Wüthrich, Manfred Wuhrer, Laurence H. Beck, David J. Salant, Gérard Lambeau, Andreas D. Kistler

×

Figure 1

Anti-PLA2R1–positive sera induce complement-dependent degradation of synaptopodin and NEPH1.

Options: View larger image (or click on image) Download as PowerPoint
Anti-PLA2R1–positive sera induce complement-dependent degradation of syn...
(A) Differentiated human podocytes infected with PLA2R1-containing lentivirus and control cells infected with empty vector were preincubated with 2.5% anti-PLA2R1–positive patient sera with high antibody titer (>1:160) for 30 minutes, treated for the indicated time periods with 5% NHS as a complement source, and analyzed by Western blotting against the indicated proteins. Bars represent mean ± SEM of the average of 3 independent experiments in each of 5 patients, *P < 0.05 by 1-way ANOVA and Tukey’s post hoc test for the comparison with t = 0. (B) Immunofluorescence analysis of synaptopodin, NEPH1, and actin fibers in the same assay with serum from a patient with an anti-PLA2R1 titer of 451 RU/mL. The representative images were captured using a Leica SP8 upright confocal microscope and 63× objective lens. (C) The results were confirmed in a large cohort of anti-PLA2R1–positive pMN patients (pMN+) with active disease or in remission (act/rem), anti-PLA2R1–negative pMN patients (pMN–), secondary membranous nephropathy (sMN), other glomerular diseases (other), and healthy controls. Shown are synaptopodin and NEPH1 levels after preincubation with serum and 60 minutes of NHS exposure. Note that 1 patient with focal segmental glomerulosclerosis (group “other”) showed a reduction of synaptopodin and NEPH1 that was not dependent on PLA2R1 expression (Supplemental Figure 10). (D) Correlation of synaptopodin and NEPH1 levels with ELISA-based anti-PLA2R1 antibody concentrations of patient sera used in the assay. r, Pearson’s correlation coefficient; *P < 0.05.